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Original Article |

An Epigenetically Derived Monoclonal Origin for Recurrent Respiratory Papillomatosis FREE

Josena Kunjoonju Stephen, MD; Lori E. Vaught, MD; Kang Mei Chen, MD; Veena Shah, MD; Vanessa G. Schweitzer, MD; Glendon Gardner, MD; Michael S. Benninger, MD; Maria J. Worsham, PhD
[+] Author Affiliations

Author Affiliations: Research Division, Department of Otolaryngology–Head and Neck Surgery (Drs Stephen, Vaught, Chen, Schweitzer, Gardner, Benninger, and Worsham), and Department of Pathology (Dr Shah), Henry Ford Hospital, Detroit, Michigan.


Arch Otolaryngol Head Neck Surg. 2007;133(7):684-692. doi:10.1001/archotol.133.7.684.
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Objective  To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis.

Design  Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay.

Subjects  Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old).

Results  Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases.

Conclusions  A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.

Figures in this Article

Recurrent respiratory (laryngeal) papillomatosis (RRP), an extremely rare condition, is characterized by benign neoplasms within the respiratory tract and can be potentially life threatening because of airway obstruction.1 Recurrent respiratory papillomatosis presents primarily as tiny or larger warts on the vocal chords. Prevalence of RRP worldwide is approximately 100 000, with 2300 new cases in the United States each year.24 Juvenile-onset disease occurs in patients from younger than 1 year to 8 years old; shows no sex difference5,6; has a rapid but often unpredictable pattern of recurrence5; tends to be a long-term, often lifelong disease; and exhibits a continuum of severity and aggressiveness. The adult form of RRP has a variable age at onset (peak, approximately 20-30 years),6,7 with a higher incidence in males.6 The severity, aggressiveness, and recurrence of the adult form tend to be less than in the juvenile form.6 Human papillomavirus types 6 and 11 account for 80% to 90% of RRP.8 A small percentage of RRP cases progress to malignancy.9

Laryngeal papillomas usually run a benign but recurrent course. Spontaneous transformation of RRP to squamous cell carcinoma is not easily characterized by a histologic progression through dysplasia over time, making these lesions difficult to diagnose histologically and clinically early in the course of the transformation of the disease.

Clonality, the property that the cells within a tumor are derived from a single parent cell, is often indicated by uniformity or relative uniformity of genetic aberrations contained within many or all cells of the tumor. Such aberrations are assumed to confer or reflect biological distinctions relevant to tumor behavior, and thus to be relevant to tumor initiation and clonal expansion.1013

Epigenetics is the regulation of changes in gene expression by mechanisms that do not involve changes in DNA sequence. Establishment and maintenance of epigenetic control (gene silencing) has several aspects, which include promoter region hypermethylation, methyl-binding proteins, DNA methyltransferases, histone deacetylases, and chromatin state. Aberrant methylation of CpG islands is a hallmark of human cancers and is found early during carcinogenesis.14 Genes in cellular pathways that are inactivated by promoter region hypermethylation include MGMT (DNA repair), p16INK4a, p15INK4b (cell cycle), DAPK (apoptosis), and GSTP1 (detoxification).15

We investigated alterations in DNA methylation in biopsy specimens of recurrences from patients with RRP to assess the contribution of promoter methylation-mediated epigenetic events in RRP tumorigenesis. Aberrant promoter methylation of 22 methylation-prone tumor suppressor genes was evaluated by means of a high-throughput multigene probe panel (41 gene probes, 35 unique genes, including control probes) in 15 RRP cases by using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay.16,17

RRP COHORT

The RRP cohort comprised 15 subjects, 3 females and 12 males, all with adult onset of respiratory papillomatosis (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old). The number of biopsy specimens from patients with recurrences ranged from 1 to 6. Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the MS-MLPA assay.

DNA EXTRACTION

As a first step, 300 μL of P-buffer (50mM Tris hydrochloride, pH 8.5; 100mM sodium chloride; 1mM EDTA; 0.5% Triton X100; 20mM dithiothreitol) was added to tubes containing whole 5-μm tissue sections or microdissected tissue. The tube was heated for 15 to 20 minutes at 90°C in a water bath and allowed to cool to 60°C. Next, 6 μL of 20-mg/mL proteinase K was added, mixed, overlaid with 3 drops of mineral oil, and spun for 5 seconds at 13 000g. This was followed by a 4- to 16-hour (overnight) incubation at 60°C. The tube was heated for 10 minutes at 90°C to denature the proteinase K and to disrupt nucleic acid formaldehyde adducts. On removal of the oil, the tube was centrifuged for 15 minutes (at 13 000g) at room temperature. Next, 250 μL of the supernatant was transferred to a clean 1.5-mL tube. On addition of 10 μL of 5M sodium chloride and 1000 mL of ethanol to the 250-μL supernatant, the tube was incubated at −20°C for least 60 minutes. This was followed by centrifugation for 15 minutes at 13 000g at −4°C. On removal of the supernatant, an additional centrifugation step for 10 seconds ensured removal of the last traces of the supernatant. Finally, the pellet was air dried and dissolved in 100 μL of double-distilled water.

MS-MLPA ASSAY

The MS-MLPA assay allows for the relative quantification of approximately 41 different DNA sequences in a single reaction requiring only 20 ng of human DNA. The standard use of the technique to observe quantitative changes in copy number has been outlined in other studies.1821 Adaptation of the MLPA to detect aberrant methylation (MS-MLPA) has been detailed elsewhere.16,17

The probe design is similar to that of ordinary MLPA probes. For 26 of 41 probes, the recognition sequence detected by the MLPA probe is contained within a restriction site for the methyl-sensitive enzyme HhaI (Figure 1).

Place holder to copy figure label and caption
Figure 1

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA).17 Probes designed to recognize HhaI sites within unmethylated regions will not generate a signal because these sequences have become cut by HhaI and cannot bind to the probe. Conversely, an MLPA probe will bind to an intact methylated site, spared by HhaI, and generate an amplification signal, producing 15 separate peaks (Figure 2) in a normal control DNA sample. Aberrant methylation is identified as the appearance of a signal peak that is otherwise absent in normal DNA samples (Figures 2, 3, and 4). CH3 indicates methyl group; PCR, polymerase chain reaction.

Graphic Jump Location

The 41-gene-probe panel (Table 1) interrogates 35 unique genes implicated in cancer, including head and neck squamous cell carcinoma, for losses and gains in a separate reaction in the absence of the methyl-sensitive enzyme HhaI. Because there are 2 probes each for VHL, CDKN2A, BRCA1, and BRCA2, and 3 probes for MLH1, a normal control DNA sample will generate 41 individual peaks in the absence of HhaI (Figure 2). A concurrently run reaction with the 41-gene-probe set in the presence of HhaI is designed to detect aberrant promoter hypermethylation by taking advantage of an HhaI site in the promoter region of 22 of the 35 unique genes (note that 1 of the 2 BRCA1 probes is designed to recognize a region outside the HhaI recognition site; Table 1). Fifteen of the 41 gene probes are designed outside an HhaI site and serve as undigested controls (Figure 2). On digestion of the sample DNA with HhaI, probes that recognize the unmethylated regions will not generate a signal because these sequences have become cut by HhaI and cannot bind to the probe (Figure 1). Conversely, an MLPA probe will bind to an intact methylated site, spared by HhaI, and generate an amplification signal (Figures 2, 3, and 4).

Place holder to copy figure label and caption
Figure 2.

Methylation-specific multiplex ligation-dependent probe amplification probe mix without (A, C) and with (B, D) HhaI enzyme (DNA sequencer, ABI 3130). Note 15 peaks in the control DNA sample (B). Presence of a peak (D) in case 12, biopsy 1, not present in the control DNA (B) is that of aberrantly methylated CDKN2B gene. bp indicates base pairs; PCR, polymerase chain reaction.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Methylation-specific multiplex ligation-dependent probe amplification probe mix without and with HhaI enzyme (DNA sequencer, ABI 3100) in a control DNA specimen (A) and 3 biopsy specimens from case 4 (B-D). Note methylation of CDKN2B in all 3 biopsy specimens and methylation of TP73 in specimens 2 and 3. bp indicates base pairs; PCR, polymerase chain reaction.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Methylation-specific multiplex ligation-dependent probe amplification probe mix without and with HhaI enzyme (DNA sequencer, ABI 3100) in a control DNA specimen (A) and 3 biopsy specimens from case 7 (B-D). Note methylation of CDKN2B in all 3 biopsy specimens and methylation of APC and GSTP1 in specimens 1 and 3. bp indicates base pairs; PCR, polymerase chain reaction.

Graphic Jump Location
Table Graphic Jump LocationTable 1. Methylation MLPA Probe Panel

In this study, approximately 20 ng of DNA was used for each of the 2 reactions, one without HhaI and the other with HhaI. Briefly, DNA from normal controls (male and female) and RRP DNA for each of the 2 MLPA reactions was diluted with water to a total volume of 5 μL. The DNA was denatured and fragmented by heating for 5 minutes at 98°C in a thermocycler. Binary MLPA probes were added and allowed to hybridize to their targets during a 16-hour incubation at 60°C. Dilution buffer and a special ligase 65 enzyme (MRC Holland, Amsterdam, the Netherlands) were added to the vial. During 15-minute incubation at 60°C, the 2 parts of a probe could be ligated to each other and become an amplifiable molecule provided that the complementary sequence was present in the sample (Figure 1). This was followed by the addition of polymerase chain reaction primers, deoxynucleotide triphosphates, and Taq polymerase, followed by the following cycles: 1 cycle of 1 minute at 95°C; 10 cycles of 30 seconds at 95°C, 30 seconds at 70°C, and 1 minute at 72°C; and 30 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 1 minute at 72°C. All (ligated) probes were amplified by the same primer pair, one of which was tagged with a fluorescent dye. The relative amounts of polymerase chain reaction product obtained reflect the relative amounts of ligated probes at the start of the polymerase chain reaction. Amplification products were analyzed on a DNA sequencer (ABI 310/ABI 3100; Applied Biosystems Inc, Foster City, California), and the products were normalized and quantified.

Aberrant methylation was identified as the appearance of a signal peak that was otherwise absent in normal DNA samples (Figures 2, 3, and 4). To quantify whether 1, 2, or more copies of a specific gene locus became aberrantly hypermethylated, a mathematical algorithm was used.17

Promoter hypermethylation was recorded in 14 of 15 cases. Of the 22 unique methylation-prone cancer genes in the multigene panel, 19 had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen (Table 2).

Table Graphic Jump LocationTable 2. Case Summary and Methylation Status

Of the 15 recurrent cases, 5 cases had at least 1 abnormally methylated gene in a recurrent biopsy, of which the CDKN2B gene showed consistent hypermethylation in all 5 cases (Table 3; Figures 2, 3, and 4). All 3 biopsy specimens in case 4 indicated hypermethylation of CDKN2B, with gain of a TP73 epigenetic event in the subsequent 2 recurrences. APC and GSTP1 were hypermethylated in 1 recurrent biopsy in case 7. In case 13, all 3 specimens showed hypermethylation of the APC and the VHL promoter regions (Table 3).

Table Graphic Jump LocationTable 3. Epigenetically Linked Recurrent Laryngeal Papilloma Cases

Dissimilar epigenetic events were noted in the remaining cases (Table 2). Aberrant methylation of CDKN2B and APC genes was most frequent, occurring in 8 of 14 cases, followed by CDKN2A in 6, TIMP3 and VHL in 5, and DAPK1, HIC1, and GSTP1 in 4 of 14 cases (Table 2).

The scientific understanding of RRP has been a slow process, and RRP remains an enigmatic disease that contributes a substantial human and financial cost to the public. Originally called juvenile laryngeal papillomatosis, the disease has been increasingly recognized in adults and is now generally referred to as recurrent respiratory papillomatosis. Histologically, RRP is a benign disease characterized by unregulated growth of wartlike neoplasms on the larynx, trachea, and bronchi. Human papillomavirus has been shown to be the cause.8 Even though a small percentage of RRP cases progress to malignancy,9 spontaneous transformation of RRP to squamous cell carcinoma does not appear to be characterized by a histologic progression through dysplasia over time,22 making these cancers difficult to diagnose histologically and clinically early in the course of the transformation of the disease.

The molecular pathogenesis of RRP implicates dysregulation of apoptosis as determined by abnormal expression of antiapoptotic factors like survivin and XIAP as contributing to papilloma growth and survival.22 Human papillomavirus 11 infection is a likely early event associated with progression of RRP to carcinoma, with increased expression of p53 and topoisomerase α-II and a reduced expression of p21(WAF1) protein gene as markers of transformation to invasive papillomatosis and squamous cell carcinoma.23,24 Additional markers of an aggressive clinical course include high S-phase fraction, proliferative index, and Ki-67 expression.25

There are few reported studies examining the molecular genetics of RRP, especially from the genomic and the epigenetics perspectives, and much remains to be learned. This study should serve as a forerunner in the delineation of genes that succumb to promoter hypermethylation silencing as underlying events in the epigenetic pathogenesis of RRP. In addition, it provides evidence of a clonal origin for RRP and permits the tracing of an epigenetic continuum implicating key tumor suppressor genes in RRP.

Epigenetic alterations produce heritable changes in gene expression without a change in the DNA coding sequence itself. Promoter region hypermethylation is known to be an early event in carcinogenesis.26,27 The consequence of CpG island hypermethylation, especially for those islands associated with tumor suppressor gene promoters is the loss of tumor suppressor gene function, which contributes to tumorigenesis.17

The most commonly methylated genes in this RRP cohort were CDKN2B and APC (8 of 14 cases), CDKN2A (6 cases), TIMP3 and VHL (5 cases), and DAPK1, HIC1, and GSTP1 (4 cases; Table 2). In addition, in the 5 RRPs with at least 1 commonly methylated gene (Table 3), CDKN2B was identified in all subsequent biopsy specimens, marking this epigenetic event as an initiating clonal alteration in the recurrence continuum in RRP.

The cyclin-dependent kinase 2A (CDKN2A) and CDKN2B genes map to 9p21 and are in tandem, with CDKN2B located 25 kilobases centromeric to CDKN2A.28,29 The CDKN2A locus controls the Rb pathway (which regulates G1/ S-phase transition) and the p53 pathway (which induces growth arrest or apoptosis in response to either DNA damage or inappropriate mitogenic stimuli) by generating 2 gene products, p16 and p14.30,31 Mutations in CDKN2A/p16 inactivate the Rb pathway, whereas deletion of the CDKN2A locus (CDKN2A/p16 and CDKN2A/p14) alter both the Rb and p53 pathways, which are important in many cancers. Inactivation of the CDKN2B/p15, CDKN2A/p14, and CDKN2A/p16 genes is a frequent event in human oral squamous cell carcinomas.17,19,32,33 The presence of aberrant methylation of p15 and p16 in precancerous oral tissues32 implicates methylation of p15 and p16 as early events in the pathogenesis of oral lesions.

Hypermethylation of the APC gene was another consistent epigenetic event in RRP, occurring in 8 of 14 cases, including biopsy specimens of recurrences in cases 7 and 13. APC (adenomatosis polyposis coli) is a tumor suppressor gene originally implicated in colon cancer. It has an important role in the Wnt signaling pathway, which is involved in the development of colorectal carcinomas. Genetic and epigenetic alterations in this gene have since been recognized in other malignant neoplasms, including oral squamous cell carcinomas, gastric cancers, and esophageal adenocarcinomas. Uesugi et al34 previously reported APC as being mutated and/or deleted in primary oral squamous cell carcinoma tissues and suggested that loss of APC function contributes to carcinogenesis in the oral region. Promoter hypermethylation is also an important mechanism of APC inactivation in oral cancers, occurring in 25% of oral squamous carcinoma cells.34

In Barrett metaplasia and dysplasia,35 hypermethylation of APC, CDKN2A, and ESR1 were usually found in a large contiguous field, suggesting either a concerted methylation change associated with metaplasia or a clonal expansion of cells with abnormal hypermethylation.

Hypermethylation of the TIMP3 and VHL genes occurred in 5 of 14 RRP cases, and of the VHL gene in all 3 biopsy specimens of case 13. The VHL gene is a tumor suppressor gene located at 3p26-p25 and is responsible for Von Hippel–Lindau syndrome, which is an inherited familial cancer syndrome that makes patients susceptible to a variety of neoplasms, malignant and benign. A study of clear-cell renal carcinomas showed that hypermethylation of VHL promoter region was associated with absence of transcript expression. It was also found that treatment of these methylated VHL tumors with a demethylating agent resulted in reexpression of the VHL transcripts.36

TIMP3 induces apoptosis,37 inhibits angiogenesis,38 impedes cell migration,39 and is a physiologic regulator of inflammation.40 Promoter methylation of TIMP3 has been observed in many tumor types41,42 and is involved in the genesis of esophageal adenocarcinoma notably during the progression from dysplasia to carcinoma.43,44

Hypermethylation of DAPK1, GSTP1, and HIC1 was less frequent, occurring in 4 of 14 cases as well as in biopsy specimens of recurrence in case 7. Death-associated protein kinase 1, DAPK1, located at 9q34.1, encodes a 160-kDa cytoskeletal-associated calcium/calmodulin-dependent serine/threonine kinase that was initially identified as a positive mediator of interferon γ–induced programmed cell death in HeLa cells.45DAPK1 expression is commonly lost in urinary bladder, breast, and B-cell neoplasms and renal cell carcinoma cell lines because of promoter hypermethylation. Aberrant promoter methylation of DAPK1 has been shown to frequently occur in human head and neck cancers,17,46 non–small-cell lung carcinomas,47 gastric and colorectal carcinomas,48,49 and uterine cervical carcinomas.50 In head and neck squamous cell carcinoma, DAPK1 promoter hypermethylation has been associated with metastasis to lymph nodes as well as advanced disease stage.46

HIC1 is a tumor suppressor gene that encodes a transcriptional repressor with 5 Kruppel-like C2H2 zinc finger motifs and an N-terminal BTB/POZ domain. Epigenetic silencing of HIC1 has been shown to significantly influence tumorigenesis.51,52 Loss or reduced HIC1 messenger RNA in pediatric tumor cell lines with aberrantly methylated HIC1 became reexpressed in all cell lines by treatment with the demethylating agent 5-aza 2′deoxycytidine.53

Glutathione S-transferase π (GSTP1) encodes for the glutathione S-transferase π enzyme, which plays an important role in detoxification. It maps to 11q1354 and also has a role in susceptibility to cancer and other diseases. Inactivation of GSTP1 by promoter hypermethylation is characteristic of corticosteroid-related neoplasms such as breast, liver, and prostate cancers.46,55 The π class of glutathione S-transferase enzymes has been associated with preneoplastic and neoplastic changes.55 Promoter hypermethylation pattern of the p16, MGMT, GSTP1, and DAPK genes have been used as molecular markers for cancer cell detection in paired serum DNA, and almost half of the patients with head and neck squamous cell carcinoma with methylated tumors were found to display these epigenetic changes in paired serum.46

Persistence of the same aberrantly methylated gene in 36% of multiple recurrent biopsy specimens (5 of 14 cases) in this study cohort supports a monoclonal origin for RRP. Neoplasia typically develops as a clonal expansion from a single cell of origin. There is ample experimental and clinical evidence favoring a monoclonal origin of cancer, and some of the strongest arguments are derived from cytogenetic investigations.10,5658

More recent approaches have used a combination of fluorescence in situ hybridization and karyotyping of tumors with chromosome rearrangements as clonal markers.11 Loss of heterozygosity patterns at different loci have also been useful as clonal markers,5961 and p53 mutations have been used as clonal markers.6264

Clonal epigenetic alterations in precancerous lesions may reflect biological peculiarities pertinent to tumor behavior. Knowledge of whether a neoplasm has a single or multiple cell origin may provide important information about its etiology and pathogenesis. The high frequency of epigenetic events characterized by consistent aberrant promoter hypermethylation of multiple tumor suppressor genes points to the use of gene silencing mechanisms as one of the driving forces behind the growth of recurrent laryngeal papillomas. Additional studies to further confirm and validate the results of this study in a larger sample are in progress.

Recurrent genomic aberrations are good indicators of genes that are causally associated with cancer development or progression. Because promoter hypermethylation is potentially reversible, the molecules that regulate the methylation status of DNA are considered promising targets for new cancer therapies. Identifying epigenetic alterations in a precancerous lesion may lead to the discovery of biomarkers that add to the knowledge of risk assessment and early detection, and may provide molecular targets for chemopreventive interventions.

Correspondence: Maria J. Worsham, PhD, Department of Otolaryngology–Head and Neck Surgery, Henry Ford Hospital, 1 Ford Pl, 1D, Detroit, MI 48202 (mworsha1@hfhs.org).

Submitted for Publication: August 1, 2006; final revision received January 22, 2007; accepted February 27, 2007.

Author Contributions: Drs Stephen and Worsham had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Stephen, Benninger, and Worsham. Acquisition of data: Vaught, Chen, Shah, Schweitzer, Gardner, and Worsham. Analysis and interpretation of data: Stephen and Worsham. Drafting of the manuscript: Stephen, Chen, Benninger, and Worsham. Critical revision of the manuscript for important intellectual content: Stephen, Shah, Schweitzer, Gardner, Benninger, and Worsham. Statistical analysis: Worsham. Obtained funding: Worsham. Administrative, technical, and material support: Chen, Shah, Schweitzer, Benninger, and Worsham. Study supervision: Stephen, Gardner, Benninger, and Worsham.

Financial Disclosure: None reported.

Funding/Support: This study was supported by R01 NIH DE 15990 from the National Institutes of Health.

Previous Presentation: This study was presented at the American Head and Neck Society 2006 Annual Meeting and Research Workshop; August 19, 2006; Chicago, Illinois.

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Baker  AHZaltsman  ABGeorge  SJNewby  AC Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro: TIMP-3 promotes apoptosis. J Clin Invest 1998;101 (6) 1478- 1487
PubMed Link to Article
Qi  JHEbrahem  QMoore  N  et al.  A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2. Nat Med 2003;9 (4) 407- 415
PubMed Link to Article
van der Laan  WHQuax  PHSeemayer  CA  et al.  Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3. Gene Ther 2003;10 (3) 234- 242
PubMed Link to Article
Mohammed  FFSmookler  DSTaylor  SE  et al.  Abnormal TNF activity in Timp3Nat Genet 2004;36 (9) 969- 977
PubMed Link to Article
Momparler  RLBovenzi  V DNA methylation and cancer. J Cell Physiol 2000;183 (2) 145- 154
PubMed Link to Article
Esteller  MCorn  PGBaylin  SBHerman  JG A gene hypermethylation profile of human cancer. Cancer Res 2001;61 (8) 3225- 3229
PubMed
Bian  YSOsterheld  MCFontolliet  CBosman  FTBenhattar  J p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus. Gastroenterology 2002;122 (4) 1113- 1121
PubMed Link to Article
Eads  CALord  RVWickramasinghe  K  et al.  Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 2001;61 (8) 3410- 3418
PubMed
Cohen  OFeinstein  EKimchi  A DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death–inducing functions that depend on its catalytic activity. EMBO J 1997;16 (5) 998- 1008
PubMed Link to Article
Sanchez-Cespedes  MEsteller  MWu  L  et al.  Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 2000;60 (4) 892- 895
PubMed
Esteller  MSanchez-Cespedes  MRosell  RSidransky  DBaylin  SBHerman  JG Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non–small cell lung cancer patients. Cancer Res 1999;59 (1) 67- 70
PubMed
Lee  TLLeung  WKChan  MW  et al.  Detection of gene promoter hypermethylation in the tumor and serum of patients with gastric carcinoma. Clin Cancer Res 2002;8 (6) 1761- 1766
PubMed
Satoh  AToyota  MItoh  F  et al.  DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers. Br J Cancer 2002;86 (11) 1817- 1823
PubMed Link to Article
Dong  SMKim  HSRha  SHSidransky  D Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Cancer Res 2001;7 (7) 1982- 1986
PubMed
Chopin  VLeprince  D Chromosome arm 17p13.3: could HIC1 be the one [in French]? Med Sci (Paris) 2006;22 (1) 54- 61
PubMed Link to Article
Rathi  AVirmani  AKHarada  K  et al.  Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms. Clin Cancer Res 2003;9 (10, pt 1) 3674- 3678
PubMed
Moscow  JATownsend  AJGoldsmith  ME  et al.  Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer. Proc Natl Acad Sci U S A 1988;85 (17) 6518- 6522
PubMed Link to Article
Esteller  MCorn  PGUrena  JMGabrielson  EBaylin  SBHerman  JG Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 1998;58 (20) 4515- 4518
PubMed
Lee  WHMorton  RAEpstein  JI  et al.  Cytidine methylation of regulatory sequences near the π-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci U S A 1994;91 (24) 11733- 11737
PubMed Link to Article
Worsham  MJCarey  TEBenninger  MS  et al.  Clonal cytogenetic evolution in a squamous cell carcinoma of the skin from a xeroderma pigmentosum patient. Genes Chromosomes Cancer 1993;7 (3) 158- 164
PubMed Link to Article
Carey  TEWorsham  MJVan Dyke  DL Chromosomal biomarkers in the clonal evolution of head and neck squamous neoplasia. J Cell Biochem Suppl 1993;17F213- 222
PubMed Link to Article
Carey  TEVan Dyke  DLWorsham  MJ Nonrandom chromosome aberrations and clonal populations in head and neck cancer. Anticancer Res 1993;13(6B)2561- 2567
PubMed
Califano  Jvan der Riet  PWestra  W  et al.  Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res 1996;56 (11) 2488- 2492
PubMed
Scholes  AGWoolgar  JABoyle  MA  et al.  Synchronous oral carcinomas: independent or common clonal origin? Cancer Res 1998;58 (9) 2003- 2006
PubMed
Partridge  MEmilion  GPateromichelakis  SPhillips  ELangdon  J Field cancerisation of the oral cavity: comparison of the spectrum of molecular alterations in cases presenting with both dysplastic and malignant lesions. Oral Oncol 1997;33 (5) 332- 337
PubMed Link to Article
el-Naggar  AKLai  SLuna  MA  et al.  Sequential p53 mutation analysis of pre-invasive and invasive head and neck squamous carcinoma. Int J Cancer 1995;64 (3) 196- 201
PubMed Link to Article
Ribeiro  USafatle-Ribeiro  AVPosner  MC  et al.  Comparative p53 mutational analysis of multiple primary cancers of the upper aerodigestive tract. Surgery 1996;120 (1) 45- 53
PubMed Link to Article
Lydiatt  WMAnderson  PEBazzana  T  et al.  Molecular support for field cancerization in the head and neck. Cancer 1998;82 (7) 1376- 1380
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA).17 Probes designed to recognize HhaI sites within unmethylated regions will not generate a signal because these sequences have become cut by HhaI and cannot bind to the probe. Conversely, an MLPA probe will bind to an intact methylated site, spared by HhaI, and generate an amplification signal, producing 15 separate peaks (Figure 2) in a normal control DNA sample. Aberrant methylation is identified as the appearance of a signal peak that is otherwise absent in normal DNA samples (Figures 2, 3, and 4). CH3 indicates methyl group; PCR, polymerase chain reaction.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Methylation-specific multiplex ligation-dependent probe amplification probe mix without (A, C) and with (B, D) HhaI enzyme (DNA sequencer, ABI 3130). Note 15 peaks in the control DNA sample (B). Presence of a peak (D) in case 12, biopsy 1, not present in the control DNA (B) is that of aberrantly methylated CDKN2B gene. bp indicates base pairs; PCR, polymerase chain reaction.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Methylation-specific multiplex ligation-dependent probe amplification probe mix without and with HhaI enzyme (DNA sequencer, ABI 3100) in a control DNA specimen (A) and 3 biopsy specimens from case 4 (B-D). Note methylation of CDKN2B in all 3 biopsy specimens and methylation of TP73 in specimens 2 and 3. bp indicates base pairs; PCR, polymerase chain reaction.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Methylation-specific multiplex ligation-dependent probe amplification probe mix without and with HhaI enzyme (DNA sequencer, ABI 3100) in a control DNA specimen (A) and 3 biopsy specimens from case 7 (B-D). Note methylation of CDKN2B in all 3 biopsy specimens and methylation of APC and GSTP1 in specimens 1 and 3. bp indicates base pairs; PCR, polymerase chain reaction.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Methylation MLPA Probe Panel
Table Graphic Jump LocationTable 2. Case Summary and Methylation Status
Table Graphic Jump LocationTable 3. Epigenetically Linked Recurrent Laryngeal Papilloma Cases

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Eads  CALord  RVKurumboor  SK  et al.  Fields of aberrant CpG island hypermethylation in Barrett's esophagus and associated adenocarcinoma. Cancer Res 2000;60 (18) 5021- 5026
PubMed
Herman  JGLatif  FWeng  Y  et al.  Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 1994;91 (21) 9700- 9704
PubMed Link to Article
Baker  AHZaltsman  ABGeorge  SJNewby  AC Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro: TIMP-3 promotes apoptosis. J Clin Invest 1998;101 (6) 1478- 1487
PubMed Link to Article
Qi  JHEbrahem  QMoore  N  et al.  A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2. Nat Med 2003;9 (4) 407- 415
PubMed Link to Article
van der Laan  WHQuax  PHSeemayer  CA  et al.  Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3. Gene Ther 2003;10 (3) 234- 242
PubMed Link to Article
Mohammed  FFSmookler  DSTaylor  SE  et al.  Abnormal TNF activity in Timp3Nat Genet 2004;36 (9) 969- 977
PubMed Link to Article
Momparler  RLBovenzi  V DNA methylation and cancer. J Cell Physiol 2000;183 (2) 145- 154
PubMed Link to Article
Esteller  MCorn  PGBaylin  SBHerman  JG A gene hypermethylation profile of human cancer. Cancer Res 2001;61 (8) 3225- 3229
PubMed
Bian  YSOsterheld  MCFontolliet  CBosman  FTBenhattar  J p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus. Gastroenterology 2002;122 (4) 1113- 1121
PubMed Link to Article
Eads  CALord  RVWickramasinghe  K  et al.  Epigenetic patterns in the progression of esophageal adenocarcinoma. Cancer Res 2001;61 (8) 3410- 3418
PubMed
Cohen  OFeinstein  EKimchi  A DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death–inducing functions that depend on its catalytic activity. EMBO J 1997;16 (5) 998- 1008
PubMed Link to Article
Sanchez-Cespedes  MEsteller  MWu  L  et al.  Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 2000;60 (4) 892- 895
PubMed
Esteller  MSanchez-Cespedes  MRosell  RSidransky  DBaylin  SBHerman  JG Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non–small cell lung cancer patients. Cancer Res 1999;59 (1) 67- 70
PubMed
Lee  TLLeung  WKChan  MW  et al.  Detection of gene promoter hypermethylation in the tumor and serum of patients with gastric carcinoma. Clin Cancer Res 2002;8 (6) 1761- 1766
PubMed
Satoh  AToyota  MItoh  F  et al.  DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers. Br J Cancer 2002;86 (11) 1817- 1823
PubMed Link to Article
Dong  SMKim  HSRha  SHSidransky  D Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Cancer Res 2001;7 (7) 1982- 1986
PubMed
Chopin  VLeprince  D Chromosome arm 17p13.3: could HIC1 be the one [in French]? Med Sci (Paris) 2006;22 (1) 54- 61
PubMed Link to Article
Rathi  AVirmani  AKHarada  K  et al.  Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms. Clin Cancer Res 2003;9 (10, pt 1) 3674- 3678
PubMed
Moscow  JATownsend  AJGoldsmith  ME  et al.  Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer. Proc Natl Acad Sci U S A 1988;85 (17) 6518- 6522
PubMed Link to Article
Esteller  MCorn  PGUrena  JMGabrielson  EBaylin  SBHerman  JG Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 1998;58 (20) 4515- 4518
PubMed
Lee  WHMorton  RAEpstein  JI  et al.  Cytidine methylation of regulatory sequences near the π-class glutathione S-transferase gene accompanies human prostatic carcinogenesis. Proc Natl Acad Sci U S A 1994;91 (24) 11733- 11737
PubMed Link to Article
Worsham  MJCarey  TEBenninger  MS  et al.  Clonal cytogenetic evolution in a squamous cell carcinoma of the skin from a xeroderma pigmentosum patient. Genes Chromosomes Cancer 1993;7 (3) 158- 164
PubMed Link to Article
Carey  TEWorsham  MJVan Dyke  DL Chromosomal biomarkers in the clonal evolution of head and neck squamous neoplasia. J Cell Biochem Suppl 1993;17F213- 222
PubMed Link to Article
Carey  TEVan Dyke  DLWorsham  MJ Nonrandom chromosome aberrations and clonal populations in head and neck cancer. Anticancer Res 1993;13(6B)2561- 2567
PubMed
Califano  Jvan der Riet  PWestra  W  et al.  Genetic progression model for head and neck cancer: implications for field cancerization. Cancer Res 1996;56 (11) 2488- 2492
PubMed
Scholes  AGWoolgar  JABoyle  MA  et al.  Synchronous oral carcinomas: independent or common clonal origin? Cancer Res 1998;58 (9) 2003- 2006
PubMed
Partridge  MEmilion  GPateromichelakis  SPhillips  ELangdon  J Field cancerisation of the oral cavity: comparison of the spectrum of molecular alterations in cases presenting with both dysplastic and malignant lesions. Oral Oncol 1997;33 (5) 332- 337
PubMed Link to Article
el-Naggar  AKLai  SLuna  MA  et al.  Sequential p53 mutation analysis of pre-invasive and invasive head and neck squamous carcinoma. Int J Cancer 1995;64 (3) 196- 201
PubMed Link to Article
Ribeiro  USafatle-Ribeiro  AVPosner  MC  et al.  Comparative p53 mutational analysis of multiple primary cancers of the upper aerodigestive tract. Surgery 1996;120 (1) 45- 53
PubMed Link to Article
Lydiatt  WMAnderson  PEBazzana  T  et al.  Molecular support for field cancerization in the head and neck. Cancer 1998;82 (7) 1376- 1380
PubMed Link to Article

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